UNWINDING AND TRANSLOCATION OF DNA BY HELICASES

DNA解旋酶几乎是DNA新陈代谢的各个方面所必需的，包括复制、修复、重组和转录。要全面了解这些基本的生化过程，就需要了解DNA解旋酶的酶机制。这项研究的目标是使用我们开发的新方法来比以前更详细地研究解旋酶催化的DNA解离。我们将研究两种病毒解旋酶作为广泛功能范围的代表。SV40T抗原来自SV40病毒，是一种复制型解旋酶，具有双六聚体的功能，以高度进行性的方式解开DNA。DDA来自噬菌体T4，参与复制起始和重组，作为单体或二聚体发挥作用，以分布的方式解开DNA。由于测量这些过程的方法不充分，人们对DNA的解离和易位还没有很好的了解。我们开发了一种用于DNA解旋的预稳态试纸法，其中观察到的猝发幅度将作为酶和底物浓度的函数进行评估。这个测试将作为活性部位滴定，对于将解离与ATP水解联系起来将是有价值的。将使用两种方法来测量在一个催化循环中解开的碱基对的数量：1)通过使用高锰酸钾对新形成的单链DNA进行化学修饰来确定在唯一碱基对位置的解离速率，以及2)通过将荧光核苷酸类似物加入DNA底物中的特定位置来测量在特定位置的解离速率。将通过测量解旋酶催化的与寡核苷酸末端结合的蛋白质的置换速度来研究单链DNA上的移位。为了确定在单链DNA上是否存在运动方向上的偏差，蛋白质块将被放置在寡核苷酸的两端。将使用化学修饰的DNA底物来探讨dsDNA在解离反应中的功能意义，以确定解旋酶在反应周期中与双链相互作用的程度。本文采用的新方法将以定量的方式定义DNA解旋酶的生化机制，这将有助于评估解旋酶在DNA代谢和疾病中的作用。

DNA helicases are required for virtually every aspect of DNA metabolism, including replication, repair, recombination and transcription. A comprehensive understanding of these essential biochemical processes requires knowledge of the enzymatic mechanisms of DNA helicases. The goal of this research is to use new methods that we have developed to investigate helicase-catalyzed DNA unwinding in greater detail than has previously been possible. We will study two viral helicases as representatives of a broad functional range of activities. SV40 T antigen, from the SV40 virus, is a replicative helicase that functions as a double hexamer and unwinds DNA in a highly processive manner. Dda, from bacteriophage T4, is involved in replication initiation and recombination, functions as a monomer or dimer, and unwinds DNA in a distributive manner. DNA unwinding and translocation are not well understood, due to inadequate methods to measure these processes. We have developed a pre-steady state assay for DNA unwinding in which the observed burst amplitude will be evaluated as a function of enzyme and substrate concentration. This assay will serve as an active site titration and will be valuable for relating unwinding to ATP hydrolysis. Two methods will be used to measure the number of base pairs unwound in a single catalytic cycle or "step size": 1) Unwinding rates at unique base pair positions will be determined by chemical modification of newly formed single-stranded DNA using potassium permanganate, and 2) A fluorescence spectroscopic method will be utilized to measure unwinding rates at unique positions by incorporating fluorescent nucleotide analogs into specific sites in DNA substrates. Translocation on ssDNA will be investigated by measuring the rate of helicase-catalyzed displacement of proteins bound to the ends of oligonucleotides. To determine whether there exists a bias in direction of movement on single-stranded DNA, the protein blocks will be placed on either end of the oligonucleotide. The functional significance of dsDNA during the unwinding reaction will be probed using chemically modified DNA substrates to determine the degree to which helicases interact with the duplex during the reaction cycle. The new methods utilized here will define the biochemical mechanisms of DNA helicases in a quantitative manner that will be useful for evaluating the role of helicases in DNA metabolism and disease.